Vaccines contain two components: antigen and adjuvant. The antigen is the molecular structure encoded by the pathogen or tumor against which the immune response is directed. To activate an antigen-specific immune response, the antigen must be presented in the appropriate immunostimulatory microenvironment. Adjuvants establish such microenvironments by stimulating the production of immune-activating molecules such as proinflammatory cytokines. Vaccine efficacy depends on the types of antigen and adjuvant, and how they are administered. Striking the right balance among these components is key to eliciting protective immunity.
Toll-like receptors (TLR) sense infection by recognizing pathogen associated molecular patterns and triggering inflammation. Therefore TLR ligands have been developed as vaccine adjuvants. The uptake of antigen and activation of TLR signaling by adjuvants are dynamic, extremely tenuous processes. Ideally, antigen-presenting cells (APC) that engulf antigen will also take up TLR ligand, resulting in upregulation of co-stimulatory molecules, secretion of inflammatory cytokines, and presentation of antigen to T cells. This is certainly the case when APCs process viral particles, which contain both TLR ligands (e.g., dsRNA) and viral proteins. However, in the case of cancer vaccines the antigen and TLR ligand have been administered in mixture. This approach can result in several theoretical outcomes at the injection site: APCs that engulf antigen alone, TLR ligand alone, or TLR ligand with antigen (the desired outcome). Thus, co-administration can create a problem of signal to noise in the resulting immune response (FIG. 2). Even when antigen and TLR ligand are engulfed by the same APC, the timing is critical. This was best demonstrated by Nierkens et al, who showed that uptake of TLR9 ligand prior to antigen significantly reduced cross presentation of antigen to CTLs relative to concurrent uptake (Nierkens S, et al., Cancer Res. 2008; 68:5390-5396). Accordingly, Ingale et al. have demonstrated that direct conjugation of TLR2 ligands to antigen by a covalent bond increased the titer of tumor-reactive IgG over 100,000 times relative to vaccination with a mixture of each component (Ingale S, et al., Nat Chem Biol. 2007; 3:663-667). Similarly, coupling antigen to TLR9 ligands increases the number of antigen-specific T cells 5 to 100 fold relative to co-administration of the two components separately (Krishnamachari Y, Salem A K. Adv Drug Deliv Rev. 2009; 61:205-217).
Imidazoquinoline is a double cyclic organic molecule that has been exploited as a vaccine adjuvant. Imiquimod is an FDA-approved immune response modifier administered as a cream on the skin for the treatment of cutaneous tumors. Imiquimod exerts its immunostimulatory effects through TLR 7 expressed on plasmacytoid dendritic cells and B cells in humans. Imiquimod treatment causes release of proinflammatory cytokines including interferonα, interferonγ, and IL-12, all of which are important for priming a robust Th1 immune response associated with anti-tumor and anti-viral activity in animals. Topical imiquimod has been used as a vaccine adjuvant with modest success in numerous studies targeting established tumors and viral infection. However the efficacy of imiquimod is restrained by relying solely on TLR7 signaling because TLR7 is not expressed in one of the most abundant professional APCs, the CD8α+TLR− myeloid dendritic cells (Edwards A D, et al., Eur J Immunol. 2003; 33:827-833), thereby limiting efficacy. For this reason other compounds have been developed by modification of imiquimod.
Resiquimod is a potent dual TLR 7 and TLR 8 ligand (Wu J J, et al., Antiviral Res. 2004; 64:79-83). Since TLR 8 is expressed in CD8α+ myeloid dendritic cells, it has overcome one of the limitations of imiquimod (Coffman R L, et al., Immunity; 33:492-503). Nonetheless, many factors have limited the efficacy of resiquimod and imiquimod. One recently identified mechanism for treatment failure is that although these drugs induce proinflammatory cytokines, they concurrently induce high levels of anti-inflammatory cytokines such as IL-10 (Gibson S J, et al., Cell Immunol. 2002; 218:74-86; and Lu H, et al., J Immunol; 184:5360-5367). Of clinical relevance, application of imiquimod cream works on the treated tumor, but not distal tumors, suggesting an impairment in systemic immunity (Lu H, et al., J Immunol; 184:5360-5367; and Gill V L, et al., Vet Comp Oncol. 2008; 6:55-64). Indeed blockade of IL-10 following imiquimod treatment was shown to result in control of treated and distal (untreated) tumors, demonstrating the clinical significance of the self-regulating cytokine response induced by currently used Imidazoquinolines. Thus, a need exists to develop novel imquidazolequinoline-based compounds that trigger a more desirable ratio of pro- to anti-inflammatory cytokines.
As noted above, a related concept that has recently become clear is triggering multiple receptors is typically better for immune stimulation and triggering additional receptors might shift the cytokine prolife to a more desirable one. Since imiquimod (exclusive TLR7 ligand) and resiquimod (dual TLR7/8) ligand prime limited immunity, it would be desirable to develop improved compounds that tap additional receptors. Finally, studies have indicated dual TLR7/8 agonists are suboptimally immunogenic unless they are directly conjugated to antigen (Kastenmuller K, et al., J Clin Invest; 121:1782-1796); thus new compounds that are amenable to conjugation should also be developed.